Imidazoquinoxaline protein tyrosine kinase inhibitors

ABSTRACT

Novel imidazoquinoxalines and salts thereof, pharmaceutical compositions containing such compounds, and methods of using such compounds in the treatment of protein tyrosine kinase-associated disorders such as immunologic disorders.

This application claims priority from provisional U.S. Application Ser.No. 60/056,797, filed Aug. 25, 1997, which provisional application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to imidazoquinoxalines and salts thereof,to methods of using such compounds in treating protein tyrosinekinase-associated disorders such as immunologic disorders, and topharmaceutical compositions containing such compounds.

BACKGROUND OF THE INVENTION

Protein tyrosine kinases (PTKs) are enzymes which, in conjuction withATP as a substrate, phosphorylate tyrosine residues in peptides andproteins. These enzymes are key elements in the regulation of cellsignaling including cell proliferation and cell differentiation. PTKscomprise, inter alia, receptor tyrosine kinases (RPTKs), includingmembers of the epidermal growth factor kinase family (e.g., HER1 andHER2), platelet derived growth factor (PDGF), and kinases that play arole in angiogenesis (Tie-2 and KDR); and, in addition, non-receptortyrosine kinases, including members of the Syk, JAK and Src (e.g. src,fyn, lyn, Lck and blk) families (see Bolen, J. B., Rowley, R. B., Spana,C., and Tsygankov, A. Y., “The src family of tyrosine protein kinases inhemopoietic signal transduction”, FASEB J., 6, 3403-3409 (1992); Ulrich,A. and Schlessinger, J., “Signal transduction by receptors with tyrosinekinase activity”, Cell, 61, 203-212 (1990); and Ihle, J. N., “The Janusprotein tyrosine kinases in hematopoetic cytokine signaling”, Sem.Immunol., 7, 247-254 (1995)).

Enhanced activity of PTKs has been implicated in a variety of malignantand nonmalignant proliferative diseases. In addition, PTKs play acentral role in the regulation of cells of the immune system. PTKinhibitors can thus impact a wide variety of oncologic and immunologicdisorders. Such disorders may be ameliorated by selective inhibition ofa certain receptor or non-receptor PTK, such as Lck, or due to thehomology among PTK classes, by inhibition of more than one PTK by aninhibitor.

A PTK of particular interest is Lck which is found in T cells where itis involved in phosphorylating key protein substrates. It is requiredfor productive antigen receptor signaling and cell activation. In theabsence of Lck activity, the T cell receptor (TCR) zeta chain is notphosphorylated, the kinase ZAP-70 is not activated, and Ca²⁺mobilization essential for T cell activation does not occur (see Weiss,A. and Littman, D. R. “Signal transduction by lymphocyte antigenreceptors”, Cell, 76, 263-274 (1994); Iwashima, M., Irving, B. A., vanOers, N. S. C., Chan, A. C., and Weiss, A., “Sequential interactions ofthe TCR with two distinct cytoplasmic tyrosine kinases”, Science, 263,1136-1139 (1994); and Chan, A. C., Dalton, M., Johnson, R., Kong, G.,Wang, T., Thoma, R., and Kurosaki, T., “Activation of ZAP-70 kinaseactivity by phosphorylation of tyrosine 493 is required for lymphocyteantigen receptor function”, EMBO J., 14, 2499-2508 (1995)). Inhibitorsof Lck are thus useful in the treatment of T-cell mediated disorderssuch as chronic diseases with an important T cell component, for examplerheumatoid arthritis, multiple sclerosis and lupus, as well as acutediseases where T cells are known to play an essential role, for exampleacute transplant rejection and delayed-type hypersensitivity (DTH)reactions.

SUMMARY OF THE INVENTION

The present invention provides imidazoquinoxaline compounds of thefollowing formula I and salts thereof, for use as protein tyrosinekinase inhibitors:

where

p is 0, 1, 2, 3 or 4;

each R₁, and R₂ and R₃, are independently selected from:

(1) hydrogen or R₆,

where R₆ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, heterocyclo, orheterocycloalkyl, each of which is unsubstituted or substituted with Z₁,Z₂ and one or more (preferably, one or two) groups Z₃;

(2) —OH or —OR₆;

(3) —SH or —SR₆;

(4) —C(O)_(q)H, —C(O)_(q)R₆, or —O—C(O)_(q)R₆, where q is 1 or 2;

(5) —SO₃H or —S(O)_(q)R₆;

(6) halo;

(7) cyano;

(8) nitro;

(9) —Z₄—NR₇R₈;

(10) —Z₄—N(R₉)—Z₅—NR₁₀R₁₁;

(11) —Z₄—N(R₁₂)—Z₅—R₆;

(12) —SiR₁₃R₁₄R₁₅;

(13) —P(O)(OR₆)₂;

(14) —CH═N—OR₆;

(15) any two groups R₁ may together be alkylene or alkenylene completinga 3- to 8-membered saturated or unsaturated ring together with thecarbon atoms to which they are attached, which ring is unsubstituted orsubstituted with Z₁, Z₂ and Z₃; or

(16) any two groups R₁ may, together with the carbons to which they areattached, form a heterocyclo group, which group is unsubstituted orsubstituted with Z₁, Z₂ and Z₃;

R₄ is oxygen, sulfur, or a single bond;

R₅ is alkyl, alkenyl, aryl, heterocyclo (preferably, heteroaryl), eachof which is unsubstituted or substituted with Z₁, Z₂ and one or more(preferably, one or two) groups Z₃;

R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂:

(1) are each independently hydrogen or R₆;

(2) R₇ and R₈ may together be alkylene or alkenylene, completing a 3- to8-membered saturated or unsaturated ring with the nitrogen atom to whichthey are attached, which ring is unsubstituted or substituted with Z₁,Z₂ and Z₃; or

(3) any two of R₉, R₁₀ and R₁₁ may together be alkylene or alkenylenecompleting a 3- to 8-membered saturated or unsaturated ring togetherwith the nitrogen atoms to which they are attached, which ring isunsubstituted or substituted with Z₁, Z₂ and Z₃;

R₁₃, R₁₄ and R₁₅ are each independently:

(1) alkyl; or

(2) phenyl;

Z₁, Z₂ and Z₃ are each independently:

(1) hydrogen or Z₆, where Z₆ is (i) alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl,alkylaryl, cycloalkylaryl, heterocyclo, or heterocycloalkyl; (ii) agroup (i) which is itself substituted by one or more of the same ordifferent groups (i); or (iii) a group (i) or (ii) which is substitutedby one or more of the following groups (2) to (16) of the definition ofZ₁, Z₂ and Z₃;

(2) —OH or —OZ₆;

(3) —SH or —SZ₆;

(4) —C(O)_(q)H, —C(O)_(q)Z₆, or —O—C(O)_(q)Z₆;

(5) —SO₃H or —S(O)_(q)Z₆;

(6) halo;

(7) cyano;

(8) nitro;

(9) —Z₄—NZ₇Z₈;

(10) —Z₄—N(Z₉)—Z₅—NZ₇Z₈;

(11) —Z₄—N(Z₁₀)—Z₅—Z₆;

(12) —Z₄—N(Z₁₀)—Z₅—H;

(13) oxo;

(14) —O—C(O)—Z₆;

(15) any two of Z₁, Z₂, and Z₃ may together be alkylene or alkenylenecompleting a 3- to 8-membered saturated or unsaturated ring togetherwith the atoms to which they are attached; or

(16) any two of Z₁, Z₂, and Z₃ may together be —O—(CH₂)_(q)—O—;

Z₄ and Z₅ are each independently:

(1) a single bond;

(2) —Z₁₁—S(O)_(q)—Z₁₂—;

(3) —Z₁₁—C(O)—Z₁₂—;

(4) —Z₁₁—C(S)—Z₁₂—;

(5) —Z₁₁—O—Z₁₂—;

(6) —Z₁₁—S—Z₁₂—;

(7) —Z₁₁—O—C(O)—Z₁₂—; or

(8) —Z₁₁—C(O)—O—Z₁₂—;

Z₇, Z₈, Z₉ and Z₁₀:

(1) are each independently hydrogen or Z₆;

(2) Z₇ and Z₈, or Z₆ and Z₁₀, may together be alkylene or alkenylene,completing a 3- to 8-membered saturated or unsaturated ring togetherwith the atoms to which they are attached, which ring is unsubstitutedor substituted with Z₁, Z₂ and Z₃; or

(3) Z₇ or Z₈, together with Z₉, may be alkylene or alkenylene completinga 3- to 8-membered saturated or unsaturated ring together with thenitrogen atoms to which they are attached, which ring is unsubstitutedor substituted with Z₁, Z₂ and Z₃; and

Z₁₁ and Z₁₂ are each independently:

(1) a single bond;

(2) alkylene;

(3) alkenylene; or

(4) alkynylene.

Compounds of the formula I and salts thereof, excluding the compounds4-methoxyimidazo[1,5-a]quinoxaline-3-carboxylic acid methyl ester and4-(2-ethoxyethoxy)imidazo[1,5-a]quinoxaline-3-carboxylic acid ethylester, are novel.

DETAILED DESCRIPTION OF THE INVENTION

The following are definitions of terms used in this specification. Theinitial definition provided for a group or term herein applies to thatgroup or term throughout the present specification, individually or aspart of another group, unless otherwise indicated.

The terms “alk” or “alkyl” refer to straight or branched chainhydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbonatoms. The expression “lower alkyl” refers to alkyl groups of 1 to 4carbon atoms.

The term “alkenyl” refers to straight or branched chain hydrocarbongroups of 2 to 10, preferably 2 to 4, carbon atoms having at least onedouble bond. Where an alkenyl group is bonded to a nitrogen atom, it ispreferred that such group not be bonded directly through a carbonbearing a double bond.

The term “alkynyl” refers to straight or branched chain hydrocarbongroups of 2 to 10, preferably 2 to 4, carbon atoms having at least onetriple bond. Where an alkynyl group is bonded to a nitrogen atom, it ispreferred that such group not be bonded directly through a carbonbearing a triple bond.

The term “alkylene” refers to a straight chain bridge of 1 to 5 carbonatoms connected by single bonds (e.g., —(CH₂)_(x)— wherein x is 1 to 5),which may be substituted with 1 to 3 lower alkyl groups.

The term “alkenylene” refers to a straight chain bridge of 2 to 5 carbonatoms having one or two double bonds that is connected by single bondsand may be substituted with 1 to 3 lower alkyl groups. Exemplaryalkenylene groups are —CH═CH—CH═CH—, —CH₂—CH═CH—, —CH₂—CH═CH—CH₂—,—C(CH₃)₂CH═CH— and —CH(C₂H₅)—CH═CH—.

The term “alkynylene” refers to a straight chain bridge of 2 to 5 carbonatoms that has a triple bond therein, is connected by single bonds, andmay be substituted with 1 to 3 lower alkyl groups. Exemplary alkynylenegroups are —C≡C—, —CH₂—C≡C—, —CH(CH₃)—C≡C— and —C≡C—CH(C₂H₅)CH₂—.

The terms “ar” or “aryl” refer to phenyl, naphthyl and biphenyl.

The terms “cycloalkyl” and “cycloalkenyl” refer to cyclic hydrocarbongroups of 3 to 8 carbon atoms.

The terms “halogen” and “halo” refer to fluorine, chlorine, bromine andiodine.

The term “unsaturated ring” includes partially unsaturated and aromaticrings.

The terms “heterocycle”, “heterocyclic” or “heterocyclo” refer to fullysaturated or unsaturated, including aromatic (“heteroaryl”) ornonaromatic cyclic groups, for example, 4 to 7 membered monocyclic, 7 to11 membered bicyclic, or 10 to 15 membered tricyclic ring systems, whichhave at least one heteroatom in at least one carbon atom-containingring. Each ring of the heterocyclic group containing a heteroatom mayhave 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atomsand/or sulfur atoms, where the nitrogen and sulfur heteroatoms mayoptionally be oxidized and the nitrogen heteroatoms may optionally bequaternized. The heterocyclic group may be attached at any heteroatom orcarbon atom of the ring or ring system.

Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl,pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl,imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl,thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl,furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl,azepinyl, hexahydroazepinyl, 4-piperidonyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone,1,3-dioxolane and tetrahydro-1,1-dioxothienyl, and the like.

Exemplary bicyclic heterocyclic groups include indolyl, benzothiazolyl,benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl,tetra-hydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl,indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl,cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (suchas furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl),dihydroisoindolyl, dihydroquinazolinyl (such as3,4-dihydro-4-oxo-quinazolinyl), dihydrobenzodioxinyl (such as2,3-dihydro-1,4-benzodioxinyl), benzodioxolyl (such as1,3-benzodioxolyl), tetrahydroquinolinyl and the like.

Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.

Where q is 1 or 2, “—C(O)_(q)H” denotes —C(O)—H or —C(O)—OH;“—C(O)_(q)R₆” or “—C(O)_(q)Z₆” denote, respectively, —C(O)—R₆ or—C(O)—OR₆, or —C(O)—Z₆ or —C(O)—OZ₆; “—O—C(O)_(q)R₆” or “—O—C(O)_(q)Z₆”denote, respectively, —O—C(O)—R₆ or —O—C(O)—OR₆, or —O—C(O)—Z₆ or—O—C(O)—OZ₆; and “—S(O)_(q)R₆” or “—S(O)_(q)Z₆” denote, respectively,—SO—R₆ or —SO₂—R₆, or —SO—Z₆ or —SO₂—Z₆.

The compounds of formula I form salts which are also within the scope ofthis invention. Reference to a compound of the formula I herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic and/orbasic salts formed with inorganic and/or organic acids and bases.Zwitterions (internal or inner salts) are included within the term“salt(s)” as used herein (and may be formed, for example, where the Rsubstituents comprise an acid moiety such as a carboxyl group). Alsoincluded herein are quaternary ammonium salts such as alkylammoniumsalts. Pharmaceutically acceptable (i.e., non-toxic, physiologicallyacceptable) salts are preferred, although other salts are useful, forexample, in isolation or purification steps which may be employed duringpreparation. Salts of the compounds of the formula I may be formed, forexample, by reacting a compound I with an amount of acid or base, suchas an equivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates (such as those formedwith acetic acid or trihaloacetic acid, for example, trifluoroaceticacid), adipates, alginates, ascorbates, aspartates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates,hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates,lactates, maleates, methanesulfonates, 2-naphthalenesulfonates,nicotinates, nitrates, oxalates, pectinates, persulfates,3-phenylpropionates, phosphates, picrates, pivalates, propionates,salicylates, succinates, sulfates (such as those formed with sulfuricacid), sulfonates (such as those mentioned herein), tartrates,thiocyanates, toluenesulfonates such as tosylates, undecanoates, and thelike.

Exemplary basic salts (formed, for example, where the R substituentscomprise an acidic moiety such as a carboxyl group) include ammoniumsalts, alkali metal salts such as sodium, lithium, and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases (for example, organic amines) such as benzathines,dicyclohexylamines, hydrabamines, N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine and the like. The basic nitrogen-containing groupsmay be quaternized with agents such as lower alkyl halides (e.g. methyl,ethyl, propyl, and butyl chlorides, bromides and iodides), dialkylsulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), longchain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides), aralkyl halides (e.g. benzyl and phenethylbromides), and others.

Prodrugs and solvates of the compounds of the invention are alsocontemplated herein. The term “prodrug”, as employed herein, denotes acompound which, upon administration to a subject, undergoes chemicalconversion by metabolic or chemical processes to yield a compound of theformula I, or a salt and/or solvate thereof. Solvates of the compoundsof formula I are preferably hydrates. Any tautomers which may exist arealso contemplated herein as part of the present invention.

All stereoisomers of the present compounds, such as those which mayexist due to asymmetric carbons on the R substituents of the compound ofthe formula I, including enantiomeric and diastereomeric forms, arecontemplated within the scope of this invention. Individualstereoisomers of the compounds of the invention may, for example, besubstantially free of other isomers, or may be admixed, for example, asracemates or with all other, or other selected, stereoisomers. Thechiral centers of the present invention can have the S or Rconfiguration as defined by the IUPAC 1974 Recommendations.

Throughout the specification, groups and substituents thereof are chosento provide stable moieties and compounds.

Preferred Compounds

Compounds of the formula I, and salts thereof, wherein one or more, andespecially all, of p, R₁, R₂, R₃, R₄, and R₅ are selected from thefollowing definitions, are preferred compounds of the present invention:

p is 0, 1 or 2;

each R₁ is independently selected from hydrogen, alkyl, alkoxy, nitro,aryl, halo, heterocyclo, —Z₄—NR₇R₈ or —Z₄—N(R₁₂)—Z₅—R₆;

R₂ is selected from hydrogen or alkyl;

R₃ is selected from hydrogen or alkyl;

R₄ is selected from oxygen, sulfur, or a single bond; and

R₅ is selected from optionally substituted aryl.

Methods of Preparation

The compounds of the formula I may be prepared by methods such as thoseillustrated in the following Schemes I to IV. Solvents, temperatures,pressures, and other reaction conditions may readily be selected by oneof ordinary skill in the art. All documents cited are incorporatedherein by reference in their entirety. Starting materials arecommercially available or readily prepared by one of ordinary skill inthe art.

The methods described herein may be carried out with starting materialsand/or reagents in solution or alternatively, where appropriate, withone or more starting materials or reagents bound to a solid support (see(1) Thompson, L. A., Ellman, J. A., Chemical Reviews, 96, 555-600(1996); (2) Terrett, N. K., Gardner, M., Gordon, D. W., Kobylecki, R.J., Steele, J., Tetrahedron, 51, 8135-8173 (1995); (3) Gallop, M. A.,Barrett, R. W., Dower, W. J., Fodor, S. P. A., Gordon, E. M., Journal ofMedicinal Chemistry, 37, 1233-1251 (1994); (4) Gordon, E. M., Barrett,R. W., Dower, W. J., Fodor, S. P. A., Gallop, M. A., Journal ofMedicinal Chemistry, 37, 1385-1401 (1994); (5) Balkenhohl, F., von demBussche-Hünnefeld, Lansky, A., Zechel, C., Angewandte ChemieInternational Edition in English, 35, 2288-2337 (1996); (6) Balkenhohl,F., von dem Bussche-Hünnefeld, Lansky, A., Zechel, C., AngewandteChemie, 108, 2436-2487 (1996); and (7) Sofia, M. J., Drugs DiscoveryToday, 1, 27-34 (1996)).

As shown in Scheme I, an appropriately substituted 2-nitro halobenzene 1can be reacted with a substituted imidazole 2 in the presence of a basesuch as sodium, potassium, or cessium carbonate, or an amine base suchas triethyl amine, diisopropylethyl amine,1,8-diazabicyclo[5.4.0]undec-7-ene (“DBU”), or the like, in anappropriate solvent to give the imidazole derivative 3 (Davey, et al.,J. Med. Chem., 34, 2671 (1991)). The reaction may also be carried out inthe presence of a copper I salt such as cuprous chloride, cuprousbromide, or cuprous iodide (Sitkina, et al., Khim Geterotskil Soed in143 (1966); Grimmett, et al., Aust. J. Chem., 32, 2203 (1979); Sugaya,et al., Synthesis, 73 (1994)). Preferred X groups in 1 are F and Cl inthe absence of a copper I salt and Br and I in the presence of a copperI salt.

The nitro group of 3 may then be reduced to provide the correspondingamine 4 by methods such as those known in the art (e.g., Hudlicky,“Reductions in Organic Chemistry”, Wiley (1984)), for example, bycatalytic hydrogenation, or by use of SnCl₂, FeCl₃, sodium dithionite,or the like.

The aminoimidazole derivative 4 may be reacted with carbonyldiimidazole5, or alternatively phosgene or phosgene equivalents, to give thequinoxolinone 6 (Davey, et al., J. Med. Chem., 34, 2671 (1991)).Quinoxolinone 6 may also exist as its tautomer 6a.

When R₂ is hydrogen a mixture of regioisomers is possible at theimidazole ring, and the desired 1,5-isomer is preferably separated fromthe 1,2-isomer by methods such as fractional crystallization, orchromatography on silica gel or C-18. It is preferred to obtain acompound of the formula I substantially free of its corresponding1,2-regioisomer.

Quinoxolinone 6 may be converted into its chloroimidate 7 in thepresence of phosphorylchloride, or analogous reagents such as SOCl₂,PCl₅, PPh₃/CCl₄, or the like, and 7 reacted with the appropriate alcoholor thiol 8 in the presence of a base such as sodium or potassiumhydride, or the like (see J. Med. Chem., 36, 2335 (1993); and Collect.Czech. Chem. Commun., 55, 2493 (1990)), or the appropriateorganometallic reagent 9 where M is an organometallic group such as aGrignard, organolithium, organotin, or boronic acid derivative, or thelike (see Tetrahedron Lett., 32, 2273 (1991); and J. Chem. Soc. C, 2376(1969)) to give compound I.

As shown in Scheme II, an appropriately substituted amino halobenzene 10may be converted to the corresponding amide 13 by either of twomethods: 1) direct coupling with the N-protected imidazole carboxylicacid 11 using peptide coupling procedures such as standard methods knownin the art (see, for example, Bodanszky, “Principles of peptidesynthesis”, Springer-Verlag (1984); Bodanszky and Bodansky, “ThePractice of Peptide Chemistry”, Springer-Verlag (1984)), followed byremoval of the Z protecting group (see, for example, Greene, “ProtectiveGroups in Organic Synthesis”, Wiley (1991)); or 2) reaction of 10 withthe dimer 12 the latter prepared by methods such as those known in theart (Kasina and Nematollahi, Synthesis, 162 (1975); Godefrol, et al., J.Org. Chem., 29, 3707 (1964)). Exemplary nitrogen protecting groupsinclude carbobenzyloxy or t-butoxycarbonyl. The dimer 12 may also beprepared by coupling the imidazole carboxylic acid:

employing thionyl chloride or oxalyl chloride, preferably in thepresence of dimethylformamide and heat.

Amide 13 may then be converted to the quinoxolinone 6 by treatment witha base such as sodium, potassium, or cessium carbonate or an amine basesuch as triethylamine, diisopropylamine, DBU, or the like; or in thepresence or absence of a copper I salt such as cuprous chloride, cuprousbromide, or cuprous iodide. Preferred X groups in 13 are F and Cl in theabsence of a copper (I) salt and Br and I in the presence of a copper(I) salt. Conversion of 6 to compound I may then be carried out asdescribed in Scheme I.

As shown in Scheme III, the protected quinoxolone 14 may be reacted withtosylmethylisocyanide (“TOSMIC”) in the presence of a base such assodium hydride, n-butyl lithium, lithium, sodium, potassium, or cessiumcarbonate, or the like, to give the imidazoquinoxolone 15 (Silvestri, etal., J. Heterocyclic Chem., 31, 1033 (1994); Massa, et al., J.Heterocyclic Chem., 30, 749 (1993)). After removal of the nitrogenprotecting group Z, conversion of 15 to compound I (R₂, R₃=H) can becarried out by methodology described in Scheme I.

The starting materials for this Scheme are commercially available or maybe readily prepared as described, for example, in 1) Japanese Patent JPO5140120; 2) Epperson, et al., Bioorg. Med. Chem. Lett., 3, 2801 (1993);3) Bekerman, et al., J. Heterocycl. Chem., 29, 129 (1992); 4)Kazimierczuk and Pfleiderer, Liebigs Ann. Chem., 754 (1982); 5) a)Kalyanam and Manjunatha, Indian J. Chem., Sect. B, 31, 415 (1992) and b)Wear and Hamilton, J. Am. Chem. Soc., 72, 2893 (1950); and 6) Sakaata,et al., Heterocycles, 23, 143 (1983).

As shown in Scheme IV, a chloro- or dichloroquinoxoline 16 may bereacted with 1 equivalent of 8 or 9 by methods described in Scheme I.Where Y=Cl in 17, treatment with an isocyanide 18 and base as describedin Scheme III gives Compound I (R₂=H) (Jacobsen, et al., J. Med. Chem.,39, 3820 (1996)). R₃ is selected such that the anion of 18 can readilyform. When R₃ is a trimethylsilyl (TMS) group, it can subsequently beremoved by protodesilylation with an acid such as trifluoracetic acid(Funk & Vollhardt, J. Am. Chem. Soc., 99, 5483 (1977)) to give compoundI (R₂, R₃=H). Alternatively, the above order of addition may be reversedwhereby isocyanide 18 is first added to 16 (Y=Cl) followed by 8 or 9.

Where Y=H in 17 TOSMIC and base may be added as described in Scheme IIIto give compound I (R₂, R₃=H).

The starting materials for this Scheme are commercially available or maybe prepared, for example, as described in Curd et al., J. Chem. Soc.,1271 (1949).

Utility

The compounds of the present invention inhibit protein tyrosine kinases,especially Src-family kinases such as Lck, Fyn, Lyn, Src, Yes, Hck, Fgrand Blk, and are thus useful in the treatment, including prevention andtherapy, of protein tyrosine kinase-associated disorders such asimmunologic disorders. “Protein tyrosine kinase-associated disorders”are those disorders which result from aberrant tyrosine kinase activity,and/or which are alleviated by the inhibition of one or more of theseenzymes. For example, Lck inhibitors are of value in the treatment of anumber of such disorders (for example, the treatment of autoimmunediseases), as Lck inhibition blocks T cell activation. The treatment ofT cell mediated diseases, including inhibition of T cell activation andproliferation, is a particularly preferred embodiment of the presentinvention. Compounds which selectively block T cell activation andproliferation are preferred. Compounds of the present invention whichblock the activation of endothelial cell PTK by oxidative stress,thereby limiting surface expression of adhesion molecules that induceneutrophil binding, and which inhibit PTK necessary for neutrophilactivation are useful, for example, in the treatment of ischemia andreperfusion injury.

The present invention thus provides methods for the treatment of proteintyrosine kinase-associated disorders, comprising the step ofadministering to a subject in need thereof at least one compound of theformula I in an amount effective therefor. Other therapeutic agents suchas those described below may be employed with the inventive compounds inthe present methods. In the methods of the present invention, such othertherapeutic agent(s) may be administered prior to, simultaneously withor following the administration of the compound(s) of the presentinvention.

Use of the compounds of the present invention in treating proteintyrosine kinase-associated disorders is exemplified by, but is notlimited to, treating a range of disorders such as: transplant (such asorgan transplant, acute transplant or heterograft or homograft (such asis employed in burn treatment)) rejection; protection from ischemic orreperfusion injury such as ischemic or reperfusion injury incurredduring organ transplantation, myocardial infarction, stroke or othercauses; transplantation tolerance induction; arthritis (such asrheumatoid arthritis, psoriatic arthritis or osteoarthritis); multiplesclerosis; inflammatory bowel disease, including ulcerative colitis andCrohn's disease; lupus (systemic lupus erythematosis); graft vs. hostdisease; T-cell mediated hypersensitivity diseases, including contacthypersensitivity, delayed-type hypersensitivity, and gluten-sensitiveenteropathy (Celiac disease); psoriasis; contact dermatitis (includingthat due to poison ivy); Hashimoto's thyroiditis; Sjogren's syndrome;Autoimmune Hyperthyroidism, such as Graves′ Disease; Addison's disease(autoimmune disease of the adrenal glands); Autoimmune polyglandulardisease (also known as autoimmune polyglandular syndrome); autoimmunealopecia; pernicious anemia; vitiligo; autoimmune hypopituatarism;Guillain-Barre syndrome; other autoimmune diseases; cancers where Lck orother Src-family kinases such as Src are activated or overexpressed,such as colon carcinoma and thymoma, or cancers where Src-family kinaseactivity facilitates tumor growth or survival; glomerulonephritis, serumsickness; uticaria; allergic diseases such as respiratory allergies(asthma, hayfever, allergic rhinitis) or skin allergies; scleracierma;mycosis fungoides; acute inflammatory responses (such as acuterespiratory distress syndrome and ishchemia/reperfusion injury);dermatomyositis; alopecia areata; chronic actinic dermatitis; eczema;Behcet's disease; Pustulosis palmoplanteris; Pyoderma gangrenum;Sezary's syndrome; atopic dermatitis; systemic schlerosis; and morphea.The present invention also provides a method for treating theaforementioned disorders such as atopic dermatitis by administration ofany compound capable of inhibiting protein tyrosine kinase.

Src-family kinases other than Lck, such as Hck and Fgr, are important inthe Fc gamma receptor induced respiratory burst of neutrophils as wellas the Fc gamma receptor responses of monocytes and macrophages. Thecompounds of the present invention inhibit the Fc gamma inducedrespiratory burst response in neutrophils, and also inhibit the Fc gammadependent production of TNF alpha in the monocyte cell line THP-1 thatdoes not express Lck. The ability to inhibit Fc gamma receptor dependentneutrophil, monocyte and macrophage responses results in additionalanti-inflammatory activity for the present compounds beyond theireffects on T cells. This activity is especially of value, for example,in the treatment of inflammatory diseases such as arthritis orinflammatory bowel disease. In particular, the present compounds are ofvalue for the treatment of autoimmune glomerulonephritis and otherinstances of glomerulonephritis induced by deposition of immunecomplexes in the kidney that trigger Fc gamma receptor responses leadingto kidney damage.

In addition, Src family kinases other than Lck, such as Lyn and Src, areimportant in the Fc epsilon receptor induced degranulation of mast cellsand basophils that plays an important role in asthma, allergic rhinitis,and other allergic disease. Fc epsilon receptors are stimulated byIgE-antigen complexes. The compounds of the present invention inhibitthe Fc epsilon induced degranulation responses, including in thebasophil cell line RBL that does not express Lck. The ability to inhibitFc epsilon receptor dependent mast cell and basophil responses resultsin additional anti-inflammatory activity for the present compoundsbeyond their effect on T cells. In particular, the present compounds areof value for the treatment of asthma, allergic rhinitis, and otherinstances of allergic disease.

The combined activity of the present compounds towards monocytes,macrophages, T cells, etc. may be of value in the treatment of any ofthe aforementioned disorders.

In a particular embodiment, the compounds of the present invention areuseful for the treatment of the aforementioned exemplary disordersirrespective of their etiology, for example, for the treatment oftransplant rejection, rheumatoid arthritis, multiple sclerosis,inflammatory bowel disease, lupus, graft v. host disease, T-cellmediated hypersensitivity disease, psoriasis, Hashimoto's thyroiditis,Guillain-Barre syndrome, cancer, contact dermatitis, allergic diseasesuch as allergic rhinitis, asthma, ischemic or reperfusion injury, oratopic dermatitis whether or not associated with PTK.

The present invention also provides pharmaceutical compositionscomprising at least one of the compounds of the formula I capable oftreating a protein tyrosine kinase-associated disorder in an amounteffective therefor, and a pharmaceutically acceptable vehicle ordiluent. The compositions of the present invention may contain othertherapeutic agents as described below, and may be formulated, forexample, by employing conventional solid or liquid vehicles or diluents,as well as pharmaceutical additives of a type appropriate to the mode ofdesired administration (for example, excipients, binders, preservatives,stabilizers, flavors, etc.) according to techniques such as those wellknown in the art of pharmaceutical formulation.

The compounds of the formula I may be administered by any suitablemeans, for example, orally, such as in the form of tablets, capsules,granules or powders; sublingually; buccally; parenterally, such as bysubcutaneous, intravenous, intramuscular, or intrasternal injection orinfusion techniques (e.g., as sterile injectable aqueous or non-aqueoussolutions or suspensions); nasally such as by inhalation spray;topically, such as in the form of a cream or ointment; or rectally suchas in the form of suppositories; in dosage unit formulations containingnon-toxic, pharmaceutically acceptable vehicles or diluents. The presentcompounds may, for example, be administered in a form suitable forimmediate release or extended release. Immediate release or extendedrelease may be achieved by the use of suitable pharmaceuticalcompositions comprising the present compounds, or, particularly in thecase of extended release, by the use of devices such as subcutaneousimplants or osmotic pumps. The present compounds may also beadministered liposomally.

Exemplary compositions for oral administration include suspensions whichmay contain, for example, microcrystalline cellulose for imparting bulk,alginic acid or sodium alginate as a suspending agent, methylcelluloseas a viscosity enhancer, and sweeteners or flavoring agents such asthose known in the art; and immediate release tablets which may contain,for example, microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and/or lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants such as those known inthe art. The present compounds may also be delivered through the oralcavity by sublingual and/or buccal administration. Molded tablets,compressed tablets or freeze-dried tablets are exemplary forms which maybe used. Exemplary compositions include those formulating the presentcompound(s) with fast dissolving diluents such as mannitol, lactose,sucrose and/or cyclodextrins. Also included in such formulations may behigh molecular weight excipients such as celluloses (avicel) orpolyethylene glycols (PEG). Such formulations may also include anexcipient to aid mucosal adhesion such as hydroxy propyl cellulose(HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methylcellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agentsto control release such as polyacrylic copolymer (e.g., Carbopol 934).Lubricants, glidants, flavors, coloring agents and stabilizers may alsobe added for ease of fabrication and use.

Exemplary compositions for nasal aerosol or inhalation administrationinclude solutions in saline which may contain, for example, benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, and/or other solubilizing or dispersing agents such asthose known in the art.

Exemplary compositions for parenteral administration include injectablesolutions or suspensions which may contain, for example, suitablenon-toxic, parenterally acceptable diluents or solvents, such asmannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodiumchloride solution, or other suitable dispersing or wetting andsuspending agents, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

Exemplary compositions for rectal administration include suppositorieswhich may contain, for example, a suitable non-irritating excipient,such as cocoa butter, synthetic glyceride esters or polyethyleneglycols, which are solid at ordinary temperatures, but liquify and/ordissolve in the rectal cavity to release the drug.

Exemplary compositions for topical administration include a topicalcarrier such as Plastibase (mineral oil gelled with polyethylene).

The effective amount of a compound of the present invention may bedetermined by one of ordinary skill in the art, and includes exemplarydosage amounts for an adult human of from about 0.1 to 100 mg/kg of bodyweight of active compound per day, which may be administered in a singledose or in the form of individual divided doses, such as from 1 to 4times per day. It will be understood that the specific dose level andfrequency of dosage for any particular subject may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the species, age, body weight, general health, sex and diet ofthe subject, the mode and time of administration, rate of excretion,drug combination, and severity of the particular condition. Preferredsubjects for treatment include animals, most preferably mammalianspecies such as humans, and domestic animals such as dogs, cats and thelike, subject to protein tyrosine kinase-associated disorders.

The compounds of the present invention may be employed alone or incombination with each other and/or other suitable therapeutic agentsuseful in the treatment of protein tyrosine kinase-associated disorderssuch as PTK inhibitors other than those of the present invention,antiinflammatories, antiproliferatives, chemotherapeutic agents, andimmunosuppressants.

Exemplary such other therapeutic agents include the following:cyclosporins (e.g., cyclosporin A), CTLA4-Ig, antibodies such asanti-ICAM-3, anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2,anti-CD3 (OKT-3), anti-CD4, anti-CD80, anti-CD86, monoclonal antibodyOKT3, agents blocking the interaction between CD40 and gp39, such asantibodies specific for CD40 and/or gp39 (i.e., CD154), fusion proteinsconstructed from CD40 and gp39 (CD40Ig and CD8gp39), inhibitors, such asnuclear translocation inhibitors, of NF-kappa B function, such asdeoxyspergualin (DSG), non-steroidal antiinflammatory drugs (NSAIDs)such as ibuprofen, steroids such as prednisone or dexamethasone, goldcompounds, antiproliferative agents such as methotrexate, FK506(tacrolimus, Prograf), mycophenolate mofetil, cytotoxic drugs such asazathiprine and cyclophosphamide, TNF-α: inhibitors such as tenidap,anti-TNF antibodies or soluble TNF receptor, and rapamycin (sirolimus orRapamune) or derivatives thereof, and the PTK inhibitors disclosed inthe following U.S. patent applications, incorporated herein by referencein their entirety: Ser. No. 60/069,159, filed Dec. 9, 1997, (AttorneyDocket No. QA202a^(*)), Ser. No. 09/897,338, filed concurrently herewithby Joel C. Barrish et al., “Imidazoquinoxaline Protein Tyrosine KinaseInhibitors”, (Attorney Docket No. QA202b), Ser. No. 60/065,042, filedNov. 10, 1997, (Attorney Docket No. QA207^(*)), and Ser. No. 60/076,789,filed Mar. 4, 1998 (Attorney Docket No. QA208^(*)). See the followingdocuments and references cited therein: Hollenbaugh, D., Douthwright,J., McDonald, V., and Aruffo, A., “Cleavable CD40Ig fusion proteins andthe binding to sgp39”, J. Immunol. Methods (Netherlands), 188(1), p. 1-7(Dec. 15, 1995); Hollenbaugh, D., Grosmaire, L. S., Kullas, C. D.,Chalupny, N. J., Braesch-Andersen, S., Noelle, R. J., Stamenkovic, I.,Ledbetter, J. A., and Aruffo, A., “The human T cell antigen gp39, amember of the TNF gene family, is a ligand for the CD40 receptor:expression of a soluble form of gp39 with B cell co-stimulatoryactivity”, EMBO J (England), 11(12), p 4313-4321 (December 1992); andMoreland, L. W. et al., “Treatment of rheumatoid arthritis with arecombinant human tumor necrosis factor receptor (p75)-Fc fusionprotein, New England J. of Medicine, 337(3), p. 141-147 (1997).

The above other therapeutic agents, when employed in combination withthe compounds of the present invention, may be used, for example, inthose amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

The following assays can be employed in ascertaining the degree ofactivity of a compound (“test compound”) as a PTK inhibitor. Compoundsdescribed in the following Examples have been tested in one or more ofthese assays, and have shown activity.

Lck Enzyme Assay

Recombinant Lck expressed as a His-tagged fusion protein in insect cellsusing a baculovirus expression system and purified by nickel affinitychromatography is incubated in kinase buffer (20 mM MOPS, pH7, 10 mMMgCl₂) in the presence of the test compound. The reaction is initiatedby the addition of substrates to the final concentration of 1 μM ATP,3.3 μCi/ml [33P] g-ATP, and 0.1 mg/ml acid denatured enolase (preparedas described in Cooper, J. A., Esch, F. S., Taylor, S. S., and Hunter,T., “Phosphorylation sites in enolase and lactate dehydrogenase utilizedby tyrosine protein kinases in vivo and in vitro”, J. Biol. Chem., 259,7835-7841 (1984)). The reaction is stopped after 10 minutes by theaddition of 10% trichloroacetic acid, 100 mM sodium pyrophosphatefollowed by 2 mg/ml bovine serum albumin. The labeled enolase proteinsubstrate is precipitated at 4 degrees, harvested onto Packard Unifilterplates and counted in a Topcount scintillation counter to ascertain theLck inhibitory activity of the test compound (activity inverselyproportional to the amount of labeled enolase protein obtained). Theexact concentration of reagents and the amount of label can be varied asneeded.

This assay is advantageous as it employs an exogenous substrate(enolase) for more accurate enzyme kinetics, and can be conducted in a96-well format that is readily automated. In addition, the His-taggedLck offers much higher production yields and purity relative to GST-Lckfusion protein.

For the preparation of recombinant Lck: Human Lck was prepared as aHis-tagged fusion protein using the Life Technologies (Gibco)baculovirus vector pFastBac Hta (commercially available) in insectcells. A cDNA encoding human Lck isolated by PCR (polymerase chainreaction) was inserted into the vector and the protein was expressedusing the methods described by the manufacturer. For the production ofLck in insect cells using baculovirus, see Spana, C., O'Rourke, E. C.,Bolen, J. B., and Fargnoli, J., “Analysis of the tyrosine kinase p56lckexpressed as a glutathione S-transferase protein in Spodopterafrugiperda cells,” Protein expression and purification, Vol. 4, p.390-397 (1993).

Cell Assays

(1) Cellular Tyrosine Phosphorylation

Jurkat T cells are incubated with the test compound and then stimulatedby the addition of antibody to CD3 (monoclonal antibody G19-4). Cellsare lysed after 4 minutes or at another desired time by the addition ofa lysis buffer containing NP-40 detergent. Phosphorylation of proteinsis detected by anti-phosphotyrosine immunoblotting. Detection ofphosphorylation of specific proteins of interest such as ZAP-70 isdetected by immunoprecipitation with anti-ZAP-70 antibody followed byanti-phosphotyrosine immunoblotting. Such procedures are described inSchieven, G. L., Mittler, R. S., Nadler, S. G., Kirihara, J. M., Bolen,J. B., Kanner, S. B., and Ledbetter, J. A., “ZAP-70 tyrosine kinase,CD45 and T cell receptor involvement in UV and H₂O₂ induced T cellsignal transduction”, J. Biol. Chem., 269, 20718-20726 (1994), and thereferences incorporated therein. The Lck inhibitors inhibit the tyrosinephosphorylation of cellular proteins induced by anti-CD3 antibodies.

For the preparation of G19-4, see Hansen, J. A., Martin, P. J., Beatty,P. G., Clark, E. A., and Ledbetter, J. A., “Human T lymphocyte cellsurface molecules defined by the workshop monoclonal antibodies,” inLeukocyte Typing I, A. Bernard, J. Boumsell, J. Dausett, C. Milstein,and S. Schlossman, eds. (New York: Springer Verlag), p. 195-212 (1984);and Ledbetter, J. A., June, C. H., Rabinovitch, P. S., Grossman, A.,Tsu, T. T., and Imboden, J. B., “Signal transduction through CD4receptors: stimulatory vs. inhibitory activity is regulated by CD4proximity to the CD3/T cell receptor”, Eur. J. Immunol., 18, 525 (1988).

(2) Calcium Assay

Lck inhibitors block calcium mobilization in T cells stimulated withanti-CD3 antibodies. Cells are loaded with the calcium indicator dyeindo-1, treated with anti-CD3 antibody such as the monoclonal antibodyG19-4, and calcium mobilization is measured using flow cytometry byrecording changes in the blue/violet indo-1 ratio as described inSchieven, G. L., Mittler, R. S., Nadler, S. G., Kirihara, J. M., Bolen,J. B., Kanner, S. B., and Ledbetter, J. A., “ZAP-70 tyrosine kinase,CD45 and T cell receptor involvement in UV and H₂O₂ induced T cellsignal transduction”, J. Biol. Chem., 269, 20718-20726 (1994), and thereferences incorporated therein.

(3) Proliferation Assays

Lck inhibitors inhibit the proliferation of normal human peripheralblood T cells stimulated to grow with anti-CD3 plus anti-CD28antibodies. A 96 well plate is coated with a monoclonal antibody to CD3(such as G19-4), the antibody is allowed to bind, and then the plate iswashed. The antibody bound to the plate serves to stimulate the cells.Normal human peripheral blood T cells are added to the wells along withtest compound plus anti-CD28 antibody to provide co-stimulation. After adesired period of time (e.g., 3 days), the [3H]-thymidine is added tothe cells, and after further incubation to allow incorporation of thelabel into newly synthesized DNA, the cells are harvested and counted ina scintillation counter to measure cell proliferation.

The following Examples illustrate embodiments of the present invention,and are not intended to limit the scope of the claims. Abbreviationsemployed in the Examples are defined below. Compounds of the Examplesare identified by the example and step in which they are prepared (forexample, “1A” denotes the title compound of step A of Example 1), or bythe example only where the compound is the title compound of the example(for example, “2” denotes the title compound of Example 2).

Abbreviations

aq.=aqueous

Boc=t-butoxycarbonyl

Bu=butyl

DMF=dimethylformamide

DMSO=dimethylsulfoxide

EtOAc=ethyl acetate

EtOH=ethanol

h=hours

hex=hexane

Me=methyl

MeOH=methanol

min.=minutes

MOPS=4-morpholine-propanesulfonic acid

MS=mass spectrometry

Ms=mesyl

n-Bu=n-butyl

Pd/C=palladium on carbon

Ph=phenyl

Ret Time=retention time

sat.=saturated

TFA=trifluoroacetic acid

THF=tetrahydrofuran

Ts=tosyl

EXAMPLE 1

Preparation of 4-Phenylimidazo[1,5-a]quinoxaline

A. 2-Quinoxalinol

To a solution of 1,2-phenylene diamine (1.98 g, 18.34 mmol) in 25 mL ofabsolute EtOH was added a solution of ethyl glyoxylate in toluene (1.1eq., 20.17 mmol, 19% w/w). The mixture was heated to reflux for 3 h.After cooling to room temperature, water (100 ml) was added. Theprecipitate was collected by filtration and washed with water, thendried under high vacuum to give 2.36 g (88%) of 1A as a beige-coloredsolid.

B. 1-[(4-Methoxyphenyl)methyl]-2(1H)-quinoxalinone

4.4 g of NaH (1.1 eq., 0.11 mol, 60% in mineral oil) was washed oncewith hexane and suspended in dry DMF (50 mL). To this suspension cooledat 0° C. was added 1A (14.6 g, 0.10 mol) in dry DMF (100 mL). Thereaction mixture was stirred at ambient temperature for 30 min. Aftercooling to 0° C., a solution of 4-methoxy benzylchloride (14.9 mL, 0.11mol) in 50 mL of dry DMF was added, followed by n-Bu₄NI (7.38 g, 0.2eq., 20 mmol). The mixture was stirred at room temperature for 2 h, thenwas heated at 60° C. for 2 h. The reaction mixture was partitionedbetween sat. NH₄Cl solution and EtOAc and extracted with EtOAc. Thecombined extracts were washed with water and dried over anhydrousNa₂SO₄. Concentration in vacuo gave a crude product as an off-whitesolid. Trituration with ether overnight gave, after filtration, 14.2 gof 1B as an off-white solid (first crop). The mother liquid wasconcentrated and chromatographed (hexane-EtOAc: 3:1 to 1:1) on silicagel to give an additional 5.51 g of 1B as an off-white solid (74%combined yield).

C. 5-[(4-Methoxyphenyl)methyl]imidazo[1,5-a]quinoxalin-4(5H)-one

1.82 g of NaH (2.2 eq., 45.52 mmol, 60% in mineral oil) was washed oncewith hexane and suspended in dry THF (40 ml). To this suspension cooledat 0° C. was added a mixture of 1B (5.51 g, 20.7 mmol) and tosylmethylisocyanide (4.44 g, 22.76 mmol) in dry THF (80 mL). The reaction mixturewas stirred at 0° C. for 30 min, warmed to room temperature and stirredfor an additional 1.0 h. The reaction mixture was poured onto a mixtureof ice-water (1.5 L). The light beige-colored precipitate was collectedby filtration and washed with water and dried under high vacuum.Trituration with MeOH and ether gave 5.72 g (94%) of 1C as a light beigesolid.

D. Imidazo[1,5-a]quinoxalin-4(5H)-one

To a solution of 1C (7.69 g, 26.22 mmol) in 130 mL of trifluoroaceticacid was added 52 mL of anisole, followed by dropwise addition of 26 mLof trifluoromethane sulfonic acid. The mixture was stirred at roomtemperature overnight. Concentration in vacuo gave a residue which wasadded carefully via pipet to a mixture of cold saturated NaHCO₃. Theresulting orange-yellow precipitate was collected by filtration andwashed first with water, then ether to give 13.5 g of crude productafter drying under high vacuum. This material was stirred in 1 L of MeOHovernight and the white precipitate was removed by filtration. Thefiltrate was concentrated in vacuo and the residue was again trituratedwith ether to give, after drying under high vacuum, 4.23 g (91%) of 1Das a light yellow solid.

1D was alternatively prepared as follows, employing as a startingmaterial 1E(ii) prepared below:

To a solution of 1E(ii) (3.3 g, 16.1 mmol) in 30 mL dimethylacetamidewas added KC₂CO₃ (4.4 g, 32.16 mmol). The reaction mixture was heated toreflux for 18 h then the reaction was concentrated in vacuo and H₂O wasadded. The solid precipitate was filtered, rinsed with water, and driedunder vacuum to give 2.49 g (84%) of 1D.

E. N-(2-Fluorophenyl)-1H-imidazole-4-carboxamide

(i) Imidazolecarbonyl dimer

To 4-imidazolecarboxylic acid (5 g, 44.6 mmol) in toluene (100 mL) wasadded DMF (700 μL) followed by thionyl chloride (10 mL). The suspensionwas heated to reflux for 1.5 hours, then cooled to room temperature. Thesolid was filtered with toluene rinse, then supsended in 80 mL CHCl₃followed by the addition of 12 mL triethylamine. The mixture was stirredfor 2 hours at room temperature, then filtered with CHCl₃ rinse. Thesolid product was dried under vacuo to give 4.07 g (97%) of the titledimer 1E(i). 1E(i) can also be prepared by the method described inKasina, S., Nematollahi, J., Synthesis, 162 (1975).

(ii) N-(2-Fluorophenyl)-1H-imidazole-4-carboxamide

To a solution of 2-fluoroaniline (2.3 mL, 23.4 mmol) in 10 mL THF wasadded a solution of sodium bis(trimethylsilyl)amide (47 mL, 47 mmol) inTHF. The mixture was heated to reflux for 0.5 h then cooled to roomtemperature. To the reaction mixture was added 1E(i) (2.2 g, 11.7 mmole)in 20 mL THF and heated to reflux for 2 h. The reaction mixture wascooled in ice, quenched with acetic acid and condensed in vacuo. Waterwas added to the residue and neutralized with NaHCO₃. The resultingsolid precipitate was filtered and washed with water followed by hexane.Drying in vacuo gave 3.5 g (80%) of 1E(ii).

F. 4-Chloroimidazo[5-a]quinoxaline

A mixture of 1D (2.16 g, 11.7 mmol) in 20 mL of phosphorus oxychloridewas heated to reflux for 16 h. The reaction mixture was thenconcentrated in vacuo and azeotroped with toluene. The residue was thencooled in an ice bath and cold water was added. The aqueous layer wasneutralized with NaHCO₃ and extracted with CH₂Cl₂. Concentration of theorganic layer under reduced pressure gave 2.0 g (84%) of 1F.

G. 4-Phenylimidazo[1,5-a]quinoxaline

A mixture of 1F (51 mg; 0.25 mmol), phenylboronic acid (61 mg; 0.50mmol), tetrakistriphenylphosphine palladium (5 mg), ethanol (0.8 mL),toluene (1 mL) and 2M sodium carbonate (0.7 mL) was stirred briskly at80° C. for 1.5 hrs. After cooling to room temperature, the reactionmixture was partitioned between EtOAc and water. The organic layer waswashed with brine, dried (MgSO₄) and concentrated. The residue waschromatographed on a 2.5×15 cm silica gel column, using EtOAc:Hex, 1:1and EtOAc as the mobile phase. The pure fractions were concentrated to aresidue that was subsequently dissolved in ˜2 mL of CH₂Cl₂. Hexane (8mL) was added and the CH₂Cl₂ was removed in vacuo. The yellow crystalsformed were filtered, rinsed with hexane and dried to afford 40 mg (66%)of the title product of this Example as a yellow crystalline solid. [mp165-167° C.; HPLC: Retention time=7.79 minutes, (UV 217 nM); YMC S-3 ODS(C-18) 6.0×150 mm; 70% B:A-100% B (A=90% H₂O/10% MeOH+0.2% H₃PO₄; B=90%MeOH/10% H₂O+0.2% H₃PO₄) linear gradient over 25 minutes.]

EXAMPLE 2

Preparation of 4-(4-Methoxyphenyl)imidazo[1,5-a]quinoxaline

Analogous to the method described in Example 1 to give 54 mg (79%) ofthe title product as light yellow needles. [mp 187-188° C.; HPLC:Retention time=7.85 minutes, HPLC conditions as described in Example 1.]

EXAMPLE 3

Preparation of 4-(4-Fluorophenyl)imidazo[1,5-a]quinoxaline

Analogous to the method described in Example 1 to give 19 mg (55%) ofthe title product as a yellow solid. [mp 214-215° C.; HPLC: Retentiontime=8.89 minutes, HPLC conditions as described in Example 1.]

EXAMPLE 4

Preparation of 4-(2,6-Dimethylphenyl)imidazo[1,5-a]quinoxaline

Analogous to the method described in Example 1 (the boronic acidemployed was prepared as described in Tetrahedron Lett., 33, 265 (1992))to give 22 mg (55%) of the title product as a white powder. [mp 115-125°C.; HPLC: Retention time=8.24 minutes, HPLC conditions as described inExample 1.]

EXAMPLE 5

Preparation of 4-(2-Bromophenoxy)imidazo[1,5-a]quinoxaline

A mixture of 1F (40 mg; 0.2 mmol), o-bromophenol (38 mg; 0.22 mmol) andpotassium carbonate (70 mg; 0.5 mmol) in 0.5 mL of DMF was heated to 60°C. for 18 hr. After cooling, the reaction mixture was partitionedbetween EtOAc and water. The organic layer was washed with 1N NaOH,water, and brine. Drying (MgSO₄), concentration and crystallization fromCH₂Cl₂/hexane afforded 57 mg (87%) of the title product as a tancrystalline solid. [mp 180-182° C.; HPLC: Retention time=12.04 minutes,HPLC conditions as described in Example 1.]

EXAMPLE 6

Preparation of 4-[(2-Bromophenyl)thio]imidazo[1,5-a]quinoxaline

A mixture of 1F (40 mg; 0.2 mmol), o-bromothiophenol (26 μL; 0.2 mmol)and potassium carbonate (70 mg; 0.5 mmol) in 0.5 mL of DMF was stirredat room temperature for 18 hr. An additional amount of bromothiophenol(13 μl; 0.1 mmol) was added and the reaction was stirred 2 hr. Thereaction mixture was partitioned between EtOAc, and water. The organiclayer was washed with 1N NaOH, water, and brine. Drying (MgSO₄) andconcentration afforded a solid residue. The residue was recrystallizedfrom CH₂Cl₂/hexane and allowed to stand at 5° C. for 18 hr. Filtrationand drying afforded 53 mg (75%) of the title product as a tancrystalline solid. [mp 191-192° C.; HPLC: Retention time=15.25 minutes,HPLC conditions as described in Example 1.]

EXAMPLE 7

Preparation of4-(2-Chloro-6-methylphenoxy)-7,8-dimethoxyimidazo[1,5-a]quinoxaline

A mixture of 4-chloro-7,8-dimethoxyimidazo[1,5-a]quinoxaline, preparedas described in U.S. application Ser. No. 09/097,338,“Imidazoquinoxaline Protein Tyrosine Kinase Inhibitors,” filed by JoelC. Barrish et al. concurrently herewith (Attorney Docket No. QA202b) (20mg, 0.076 mmol), potassium carbonate (52 mg, 0.38 mmol) and2-chloro-6-methylphenol (13 mg, 0.091 mmol) in 0.5 mL of dry DMF washeated at 80° C. overnight. Upon cooling, the solvent was removed underreduced pressure and the residue was purified by preparative HPLC togive 9.0 mg of the title compound as a tan solid. HPLC Ret. time=4.075min (HPLC Conditions: 0% B to 100% B; 4 minute gradient; 2 minute hold;4 mL/min.; 254 nm; YMC S5 C18 Rapid Resolution column 4.6×50 mm; A: 90%H₂O−10% MeOH−0.2% H₃PO₄; B: 10% H₂O−90% MeOH−0.2% H₃PO₄).

What is claimed is:
 1. A method for the treatment of T-cell mediated disorders, comprising the step of administering to a subject in need thereof an effective amount of an Lck inhibitor of the following formula I or a pharmaceutically acceptable salt thereof:

where p is 0, 1, 2, 3 or 4; each R₁, and R₂ are independently selected from: (1) hydrogen or R₆, where R₆ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, heterocyclo, or heterocycloalkyl, each of which is unsubstituted or substituted with Z₁, Z₂ and one or more groups Z₃; (2) —OH or —OR₆; (3) —SH or —SR₆; (4) —C(O)_(q)H, —C(O)_(q)R₆, or —O—C(O)_(q)R₆, where q is 1 or 2; (5) —SO₃H or —S(O)_(q)R₆; (6) halo; (7) cyano; (8) nitro; (9) —Z₄—NR₇R₆; (10) —Z₄—N(R₉)—Z₅—NR₁₀R₁₁; (11) —Z₄—N(R₁₂)—Z₅—R₆; (12) —SiR₁₃R₁₄R₁₅; (13) —P(O)(OR₆)₂; (14) —CH═N—OR₆; (15) any two groups R₁ may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the carbon atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; or (16) any two groups R₁ may, together with the carbons to which they are attached, form a heterocyclo group, which group is unsubstituted or substituted with Z₁, Z₂ and Z₃; R₃ is (1) hydrogen or R₆, where R₆ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, or heterocycloalkyl, each of which is unsubstituted or substituted with Z₁, Z₂ and one or groups Z₃; (2) —OH or —OR₆; (3) —SH or —SR₆; (4) —C(O)H, —C(O)R₆, or —C(O)R₆; (5) —SO₃H or —S(O)_(q)R₆ where q is 1 or 2; (6) halo; (7) cyano; (8) nitro; (9) —Z₄—NR₇R₈; (10) —Z₄—N(R₉)—Z₅—NR₁₀R₁₁; (11) —Z₄—N(R₁₂)—Z₅—R₆; (12) —SiR₁₃R₁₄R₁₅; (13) —P(O)(OR₆)₂; or (14) —CH═N—OR₆; R₄ is oxygen, or sulfur; R₅ is alkenyl, aryl, or aromatic heterocyclo, each of which is unsubstituted or substituted with Z₁, Z₂ and one or more groups Z₃; R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂: (1) are each independently hydrogen or R₆; (2) R₇ and R₈ may together be alkylene or alkenylene, completing a 3- to 8-membered saturated or unsaturated ring with the nitrogen atom to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; or (3) any two of R₉, R₁₀ and R₁₁ may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; R₁₃, R₁₄ and R₁₅ are each independently: (1) alkyl; or (2) phenyl; Z₁, Z₂ and Z₃ are each independently: (1) hydrogen or Z₆, where Z₆ is (i) alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, alkylaryl, cycloalkylaryl, heterocyclo, or heterocycloalkyl; (ii) a group (i) which is itself substituted by one or more of the same or different groups (i); or (iii) a group (i) or (ii) which is substituted by one or more of the following groups (2) to (16) of the definition of Z₁, Z₂ and Z₃; (2) —OH or —OZ₆; (3) —SH or —SZ₆; (4) —C(O)_(q)H, —C(O)_(q)Z₅, or —O—C(O)_(q)Z₆; (5) —SO₃H or —S(O)_(q)Z₆; (6) halo; (7) cyano; (8) nitro; (9) —Z₄—NZ₇Z₈; (10) —Z₄—N(Z₉)—Z₅—NZ₇Z₈; (11) —Z₄—N(Z₁₀)—Z₅—Z₆; (12) —Z₄—N(Z₁₀)—Z₅—H; (13) oxo; (14) —O—C(O)—Z₆; (15) any two of Z₁, Z₂, and Z₃ may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached; or (16) any two of Z₁, Z₂, and Z₃ may together be —O—(CH₂)_(q)—O—; Z₄ and Z₅ are each independently: (1) a single bond; (2) —Z₁₁—S(O)_(q—Z) ₁₂—; (3) —Z₁₁—C(O)—Z₁₂—; (4) —Z₁₁—C(S)—Z₁₂—; (5) —Z₁₁—O—Z₁₂—; (6) —Z₁₁—S—Z₁₂—; (7) —Z₁₁—O—C(O)—Z₁₂—; or (8) —Z₁₁—C(O)—O—Z₁₂—; Z₇, Z₈, Z₉ and Z₁₀: (1) are each independently hydrogen or Z₆; (2) Z₇ and Z₈, or Z₆ and Z₁₀, may together be alkylene or alkenylene, completing a 3- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; or (3) Z₇ or Z₈, together with Z₉, may be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; and Z₁₁ and Z₁₂ are each independently: (1) a single bond; (2) alkylene; (3) alkenylene; or (4) alkynylene; wherein the term “heterocyclo” refers to fully saturated or unsaturated, including aromatic or nonaromatic cyclic groups, selected from 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring where each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized and wherein the heterocyclic group may be attached at any available heteroatom or carbon atom of the ring or ring system.
 2. The method of claim 1 wherein said T-cell mediated disorder is a chronic disease with an important T-cell component.
 3. The method of claim 2 wherein said disease is rheumatoid arthritis.
 4. The method of claim 2 wherein said disease is multiple sclerosis.
 5. The method of claim 2 wherein said disease is lupus.
 6. The method of claim 1 wherein said T-cell mediated disorder is an acute disease where T-cells play an essential role.
 7. The method of claim 6 wherein said disease is acute transplant rejection.
 8. The method of claim 6 wherein said disease is delayed-type hypersensitivity reactions.
 9. A method for the treatment of T-cell mediated disorders, comprising the step of administering to a subject in need thereof an effective amount of an Lck inhibitor of the following formula I or a pharmaceutically acceptable salt thereof:

where p is 0, 1, 2, 3 or 4; each R₁, and R₂ are independently selected from: (1) hydrogen or R₆, where R₆ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, heterocyclo, or heterocycloalkyl, each of which is unsubstituted or substituted with Z₁, Z₂ and one or more groups Z₃; (2) —OH or —OR₆; (3) —SH or —SR₆; (4) —C(O)_(q)H, —C(O)_(q)R₆, or —O—C(O)_(q)R₆, where q is 1 or 2; (5) —SO₃H or —S(O)_(q)R₆; (6) halo; (7) cyano; (8) nitro; (9) —Z₄—NR₇R₆; (10) —Z₄—N(R₉)—Z₅—NR₁₀R₁₁; (11) —Z₄—N(R₁₂)—Z₅—R₆; (12) —SiR₁₃R₁₄R₁₅; (13) —P(O)(OR₆)₂; (14) —CH═N—OR₆; (15) any two groups R₁ may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the carbon atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; or (16) any two groups R₁ may, together with the carbons to which they are attached, form a heterocyclo group, which group is unsubstituted or substituted with Z₁, Z₂ and Z₃; R₃ is (1) hydrogen or R₆, where R₆ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, or heterocycloalkyl, each of which is unsubstituted or substituted with Z₁, Z₂ and one or groups Z₃; (2) —OH or —OR₆; (3) —SH or —SR₆; (4) —C(O)H, —C(O)R₆, or —O—C(O)R₆; (5) —SO₃H or —S(O)_(q)R₆ where q is 1 or 2; (6) halo; (7) cyano; (8) nitro; (9) —Z₄—NR₇R₈; (10) —Z₄—N(R₉)—Z₅—NR₁₀R₁₁; (11) —Z₄—N(R₁₂)—Z₅—R₆; (12) —SiR₁₃R₁₄R₁₅; or (13) —P(O)(OR₆)₂; R₄ is oxygen, sulfur, or a single bond; R₅ is aryl, or heterocyclo, each of which is unsubstituted or substituted with Z₁, Z₂ and one or more groups Z₃; R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂: (1) are each independently hydrogen or R₆; (2) R₇ and R₈ may together be alkylene or alkenylene, completing a 3- to 8-membered saturated or unsaturated ring with the nitrogen atom to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; or (3) any two of R₉, R₁₀ and R₁₁ may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; R₁₃, R₁₄ and R₁₅ are each independently: (1) alkyl; or (2) phenyl; Z₁, Z₂ and Z₃ are each independently: (1) hydrogen or Z₆, where Z₆ is (i) alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, alkylaryl, cycloalkylaryl, heterocyclo, or heterocycloalkyl; (ii) a group (i) which is itself substituted by one or more of the same or different groups (i); or (iii) a group (i) or (ii) which is substituted by one or more of the following groups (2) to (16) of the definition of Z₁, Z₂ and Z₃; (2) —OH or —OZ₆; (3) —SH or —SZ₆; (4) —C(O)_(q)H, —C(O)_(q)Z₆, or —O—C(O)_(q)Z₆; (5) —SO₃H or —S(O)_(q)Z₆; (6) halo; (7) cyano; (8) nitro; (9) —Z₄—NZ₇Z₈; (10) —Z₄—N(Z₉)—Z₅—NZ₇Z₈; (11) —Z₄—N(Z₁₀)—Z₅—Z₆; (12) —Z₄—N(Z₁₀)—Z₅—H; (13) oxo; (14)—O—C(O)—Z₆; (15) any two of Z₁, Z₂, and Z₃ may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached; or (16) any two of Z₁, Z₂, and Z₃ may together be —O—(CH₂)_(q)—O—; Z₄ and Z₅ are each independently: (1) a single bond; (2) —Z₁₁—S(O)_(q)—Z₁₂—; (3) —Z₁₁—C(O)—Z₁₂—; (4) —Z₁₁—C(S)—Z₁₂—; (5) —Z₁₁—O—Z₁₂—; (6) —Z₁₁—S—Z₁₂—; (7) —Z₁₁—O—C(O)—Z₁₂—; or (8) —Z₁₁—C(O)—O—Z₁₂—; Z₇, Z₈, Z₉ and Z₁₀: (1) are each independently hydrogen or Z₆; (2) Z₇ and Z₈, or Z₆ and Z₁₀, may together be alkylene or alkenylene, completing a 3- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; or (3) Z₇ or Z₈, together with Z₉, may be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Z₁, Z₂ and Z₃; and Z₁₁ and Z₁₂ are each independently: (1) a single bond; (2) alkylene; (3) alkenylene; or (4) alkynylene; wherein the term “heterocyclo” refers to fully saturated or unsaturated, including aromatic or nonaromatic cyclic groups, selected from 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring where each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized and wherein the heterocyclic group may be attached at any available heteroatom or carbon atom of the ring or ring system.
 10. The method of claim 9 wherein said T-cell mediated disorder is a chronic disease with an important T-cell component.
 11. The method of claim 10 wherein said disease is rheumatoid arthritis.
 12. The method of claim 10 wherein said disease is multiple sclerosis.
 13. The method of claim 10 wherein said disease is lupus.
 14. The method of claim 9 wherein said T-cell mediated disorder is an acute disease where T-cells play an essential role.
 15. The method of claim 14 wherein said disease is acute transplant rejection.
 16. The method of claim 14 wherein said disease is delayed-type hypersensitivity reactions. 